| Natural Killer T (NKT) cells are characterized by their expression of aninvariant T cell receptor (TCR), Vα14Jα18paired with Vβ8.2, Vβ7, or Vβ2in mice.NKT cells can be classified into three different functional subtypes, interleukin(IL)-4-producing, IL-17A-producing, and interferon (IFN)-γ-producing NKT cells.These NKT cells bridge the innate and acquired immune systems to mediateeffective and augmented responses. However, the limited number of NKT cells invivo hampers their analysis. To overcome this problem, the induced pluripotent stemcell (iPSC) technology is potentially a very powerful tool for analysis andapplication of NKT cells. Another important issue in NKT cell biology is tounderstand the molecular mechanisms of their fate determination. It is of interest todetermine whether TCRαor TCRβchain gene rearrangements are involved in cellfate determination in vivo, particularly in NKT cells.Here, two lines of induced pluripotent stem cell-derived mice(NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells,where one carries a rearranged Vα14Jα18on both alleles with the TCR Vβloci ingermline configuration (Vα14/WTVβmice) and the other harbors rearrangements ofboth Vα14Jα18and Vβ7on both chromosomes (Vα14/Vβ7mice).The analysis of NKT-iPSC-derived mice showed significant increased numbersand percentages ofα-GalCer/CD1d+NKT cells in the thymus, spleen and livercompared to B6mice in both Vα14/WTVβand Vα14/Vβ7mice. A similar bias wasseen in the Vβrepertoire of NKT cells from Vα14/WTVβmice, while the NKT cellsin Vα14/Vβ7mice were monoclonal and all cells exclusively used Vβ7. Genes andmolecules important for NKT cell development, including CD1d and SLAM family receptor Ly108(encoded by Slamf6), SAP, Fyn, c-Myb, c-Myc, Runx1, HEB, andEgr2, were expressed at normal levels in the thymic CD4/CD8double-positive (DP)populations in NKT-iPSC-derived mice.We also investigated development of other thymocyte subsets in the NKT-iPSCmice. NKT-iPSC-derived mice showed normal development of other immune cellsexcept for the absence ofγδT cells and disturbed development of conventional CD4αβT cells. genes important for the generation of T cells by secondary rearrangementevents, such as RAG, RORγt, the anti-apoptotic factors Bcl-xL, and Bcl-2(encodedby Rag1, Rag2, Rorc, Bcl2l1, and Bcl2, respectively), were highly expressed by DPthymocytes in both Vα14/WTVβand Vα14/Vβ7mice.The expression of CD4and IL-17RB was investigated to define the frequencyand absolute number of NKT cell subtypes in various tissues of theNKT-iPSC-derived mice. The number and frequency of the three subsets of NKTcells, CD4-IL-17RB+, CD4+IL-17RB+and IL-17RB-NKT cells corresponding toIL-17A, IL-4and IFN-γproducing NKT cell subsets, respectively, are all increasedin both Vα14/WTVβand Vα14/Vβ7mice.To investigate functional properties of the NKT cell subtypes inNKT-iPSC-derived mice, cytokine profiles were analyzed by intracellular staining todetect IFN-γ-, IL-4-and IL-17A-expressing NKT cells after activation with phorbol12-myristate13-acetate (PMA) and ionomycin in vitro. The number and frequencyof all functional NKT cell subsets in the Vα14/WTVβmice are high in all tissues,and they are functionally mature in terms of their production of IL-17A, IL-4andIFN-γcytokines, while those in Vα14/Vβ7mice, particularly the IL-17A and IL-4producing NKT cell subsets, are significantly lower in the thymus but increased inspleen and live.Consistently, thymic NKT cells from Vα14/Vβ7mice are phenotypicallyimmature based on higher expression levels of CD24and CD62L, markers of veryimmature NKT cells and naive conventional T cells, whereas these markers aredown-regulated in the peripheral NKT cells in Vα14/Vβ7mice. These results suggest that the NKT-iPSC-derived mice are a better model forNKT cell development and function study and also that the presence of apre-rearranged Vα14Jα18in the natural chromosomal context disposes thedevelopmental fate of NKT cells. |
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